Question

What features of the binding site hinder oxygen binding to Hb? What feature of the binding site facilitates oxygen binding to Hb? Explain the Bohr effect using the theory of allosteric modulation of oxygen binding to hemoglobin.

Answer 1

Haemoglobin exists in 2 conformations. One which binds oxygen with high affinity called the active or oxygen-loading state and the other which binds oxygen at 500 fold lower affinity called the inactive.

p50 is the partial pressure of oxygen which is required to saturate 50% of haemoglobin. So increasing p50 would suggest that the affinity of hemoglobin for oxygen is decreasing - it takes more oxygen to achieve 50% saturation.

The following physiological factors that influence the affinity of hemoglobin for oxygen:

1. Partial pressure of CO₂

2. pH, independent of CO₂

3. Concentration of 2,3-DPG inside the erythrocytes

4. Presence of unusual haemoglobin species

5. Temperature

Features of the binding site hinder oxygen binding to Hb - decreased oxygen affinity:

1. acidosis

2. increased pCO₂

3. increased 2,3-DPG ; 2,3-DPG = 2,3-diphophoglycerate which is a byproduct of the pathway of glycolysis.

4. Hyperthermia

5. Sulfhaemoglobin

Features of the binding site facilitate oxygen binding to Hb - increased oxygen affinity:

1. alkalosis

2. decreased pCO₂

3. decreased 2,3-DPG

4. Hypothermia

5. methaemoglobin

6. carboxyhaemoglobin

7. foetal haemoglobin

Bohr effect (using the theory of allosteric modulation of oxygen binding to haemoglobin):

The effect of decreasing pH (more hydrogen ion activity) on haemoglobin is to stabilise the deoxygenated form, decreasing its affinity for oxygen. This (together with the effects of pCO2) is the principle of Bohr effect.

deoxyhemoglobin + oxygen oxyhemoglobin + H⁺

The chemical basis for the effect of pH on the oxygen affinity of haemoglobin lies in the amino termini and side chains of two histidine molecules, histidine 146 on the β-subunit and histidine 122 on the α-subunit.

• Increasing pH stimulates hemoglobin binding to oxygen. Binding of oxygen to deoxyhemoglobin leads to the release of protons bound by the N-terminal aminogroups of theα-subunits andthe C-terminal His of the β-subunits
• As the pH decreases,dissociation of O₂ from hemoglobin is enhanced (ie the T-state is stabilized)
• Actively metabolizing tissues produce acid promoting O₂ release
• Amino groups at the N-terminal ends of the α-subunits–Implicated in binding protons.–The pKa of these groups are ~8.0.–Decreasing the pH will increase the relative positive charge, increasing the stabilityof the salt bridge and thus the T-state.•

Histidine146 is also believed to be involved in the Bohr effect.–Theoretical pKa of ~ 6.5.–Decreasing the pH will increase the relative positive charge on the histidine,increasing the stability of the salt bridge and thus the T-state